About 17.3 million Americans suffer from asthma, which is a chronic inflammatory disorder of the airways. In susceptible individuals, this inflammation causes recurrent episodes of coughing, wheezing, chest tightness and difficult breathing. Inflammation makes the airways sensitive to indoor allergens such as those derived from cockroaches, dust mites and animal dander. Asthma is also the most common chronic childhood illness, particularly in inner-city homes, and is the leading cause of children's emergency room use, hospital admission and school absence. Chronic asthma in children is highly associated with chronic respiratory disease in adulthood and has a huge health, economical and societal impact.
An extensive study was carried out by the National Cooperative Inner-City Asthma Groups, evaluating over 1500 children from eight major cities in the United States for the contribution of various indoor allergens to asthma. It was reported that of the three major indoor allergens, cockroach allergens, dust mites and animal dander, only allergens from German cockroaches showed a positive correlation with the incidence of asthma. (See Rosenstreich et al., New England J. of Med., 336 (19): 1356–1363, 1997)
Cockroach allergens are protein molecules found in their fecal pellets, dead body parts, shed cuticles and egg cases, and secretions that, when aerosolized and inhaled, trigger IgE-mediated allergic reactions to produce asthma and/or related respiratory conditions or diseases. Fumigating homes with pesticides or using baits containing toxic chemicals may initially have appeared to reduce the number of cockroaches due to the return of poisoned cockroaches to their nesting places, usually cracks and crevices, to die. But in reality, this did not reduce the indoor allergen level because the cockroaches' dead body parts, fecal pellets, and associated allergens still remained in treated homes and later became airborne and circulated in the homes. Aside from leaving toxic residues in the treated homes, the use of chemical insecticides may actually increase the allergen burden by speeding up the decaying and aerosolization processes. Cockroach allergens were also reported to be exceptionally stable and remained active for at least five years after they were produced.
Preventing the development of asthma is a major goal of current research. To achieve this goal, both the cockroaches and their associated allergens need to be removed. Efficient attractant and pheromone-based sticky traps are commercially available for catching the cockroaches. In order to eliminate the allergens produced by cockroaches, one has to know not only the exact locations of the contaminated areas but also the severity of the contamination. Currently available allergen determination procedures have failed to address these issues.
Current procedures for allergen level determination involves collecting dust samples from small arbitrary chosen areas (usually 1 m2), typically from the kitchen and/or living room, vacuuming the areas for about two minutes to collect the dust samples for analysis, then sending the dust samples to a qualified laboratory for testing as further described below. (See R. G. Hamilton, Methods: A companion to Methods in Enzymology, 13, 25–32, 1997) This procedure is based on the assumptions that aerosolized allergens will settle down as dust and the cockroaches will also deposit allergens in these areas. As indicated earlier, the source of the cockroach allergens comes from their decayed fecal pellets, decayed dried body parts, decayed shed cuticles and egg cases, and secretions which are usually found in their nesting places, such as cracks and crevices, and these are not easily accessible for detection and cleaning. Thus, currently used sampling and testing methods are not suitable for finding the locations of the cockroach nesting places and identifying other areas of intense infestation.
Aqueous extracts of ground cockroach whole body have been used for allergy testing. At least six different protein allergens from German cockroach (Blatella gemanica) whole body have either been purified or cloned. (See Arruda et al., The J. of Biol. Chem., 270 (33), 19563–19568, 1995; 270 (52), 31196–31201, 1995; and 272 (33), 20907–20912, 1997; U.S. Pat. No. 5,869,288) They are called Bla g 1 through Bla g 6, according to the order of their discovery. Currently, only indirect methods utilizing tagged secondary antibodies (for example, Enzyme-Linked Immunosorbent Assay (ELISA)) are used to quantify one or two cockroach allergens (Bla g 1 and/or Bla g 2). Furthermore, because there are so many allergens produced by cockroaches, the amount of Bla g 1 and/or Bla g 2 may not be proportional to the total allergen level at all, and the distribution of Bla g 1 and Bla g 2 among the various sources of allergens is simply not known and is highly variable from site to site. Additionally, these methods are very expensive because they require specific monoclonal antibodies against either Bla g 1 or Bla g 2 and can only be performed in a limited number of laboratories. These methods are also very time-consuming, usually requiring 2 days to complete the allergen level determination. Therefore, it is highly impractical for homeowners, pest control operators, healthcare professionals, small business owners (such as restaurant owners), etc. to collect the dust samples from arbitrarily chosen areas, mail them to the laboratory, and wait one or two weeks for the results to come back.
Cockroach allergen studies reported so far used mainly extracts from ground live cockroaches (the so-called whole body extract). Applicants have found that indoor cockroach allergens are not present in nor derived directly from live cockroaches per se as commonly perceived, but rather from their decayed fecal pellets, decayed dried dead body parts, decayed shed cuticles, and secretions (collectively called the “frass”). As the decay process occurs, the breakdown of the frass components produces the protein allergens discussed above which, when aerosolized and circulating in an indoor environment, enter the respiratory system and trigger the IgE-mediated immune response that leads to allergy and/or asthma.
Further, applicants have unexpectedly discovered that two very stable enzymes, endochitinase and exochitinase (each referred to herein as a “chitinase”), are associated with the cockroach allergens collected from both the dust samples and all cockroach frass components (decayed fecal pellets, decayed dried body parts, egg cases and cuticles, and secretions). As will be illustrated in the following Examples, the level of either one or both of these two enzymes has been found to be proportional to the total cockroach allergen level present in either the frass or the dust samples, thus making chitinases ideal markers for measurement of the total indoor cockroach allergen level in these samples. Thus, the determination of total allergen level may be made by any technique suitable for chitinase detection.
These markers are enzymes or enzyme complexes involved in the hydrolysis of the chitin molecule. Chitin, an insoluble linear β-1, 4-linked polymer of N-acetyl-β-D-glucosamine (NAG), is present in all arthropods, yeast, most fungi and some stages of nematodes. Insect chitins are usually hydrolyzed by a combination of endochitinase, which randomly cleaves the chitin molecule internally to generate predominately NAG dimers (together with some trimers and tetramers) and exochitinase (also referred to as N-acetyl-β-glucosaminidase) to generate NAG monomers. To speed up the degradation of chitin, some organisms may additionally contain a different form of endochitinase called chitobiosidase which generates exclusively NAG dimers.
Applicants have discovered that when one or both of these chitinase enzymes are present in a sample, as hereinafter defined, from an environment or site in which cockroaches are present or are believed to be present, the presence of cockroach allergens in such samples and/or the level thereof, may be immediately measured by contacting the sample with a substrate composition comprising a chitinase substrate that undergoes a measurable change of a property, such as a change in color or fluorescence, when reacted with a chitinase. The change of the property is due to a physical transformation or chemical reaction, such as hydrolysis, between the chitinase in the sample and the chitinase substrate. The magnitude of the measurable change of the property can be immediately measured, either visually or with instruments such as a fluorometer, as an indirect measurement of the total cockroach allergen level in the samples. This method for determining the total cockroach allergen levels in environments in which cockroaches may be present, as described below, can be practiced not only directly on-site but also rapidly, inexpensively, and without the cost or delays inherent in currently available test procedures, for example, the ELISA test procedure. Thus, the method of the present invention relies on the newly discovered correlation between the stable chitinase enzyme level and the total allergen level, and is thus adapted to any technique suitable for chitinase detection, while the methods of the prior art require the difficult direct determination of only one or two specific allergens from live cockroaches. The present invention not only addresses the deficiencies of the current allergen detection methods but also provides a simple alternative method that can be easily practiced on-site (for example by homeowners, healthcare professionals, pest control operators, etc.) without the cost or delays inherent in currently available test procedures, for example, the ELISA test procedure.